Cooperative Retention Mechanism In Mixed-Mode Chromatography

High-performance liquid chromatography(HPLC)represents one of the most important parts of modern analytical chemistry and has been widely used in the fields of chemistry,biology,pharmaceutical and environmental sciences.In recent years,single-mode chromatography has been rapidly developed due to its high sensitivity,selectivity and separation ability,however,it has limitations when applied in complex mixture separation.For example,reversed-phase chromatography(RPLC)is by far the most popular separation mode owing to its high selectivity and sensitivity for hydrophobic compounds,however,it fails to retain strongly polar and ionic solutes,and the separation of basic compounds often exhibits tailed peaks,poor efficiency,and irreproducible retention.Ion exchange chromatography(IEC)is frequently used for the separation of ionic and ionized substances,but,suffers from the lack of selectivity because the pure ionic interactions are relatively insensitive to the hydrophobic parts of the molecule.Ion-pair chromatography(IPC)is one of the most important methods for the simultaneous separation of neutral and ionic compounds,where the presence of an ion-pair agent in the eluent is mandatory to enhance the retention of ionic analytes.However,the use of ion-pair agents makes it incompatible with mass sensitive detection,gradient elution and preparation purification,which limit its wide application.Mixed-mode chromatography(MMC)can realize different separation modes on a single column based on multiple interaction mechanisms between the stationary phase and the solute,and simultaneously perform the separation of various samples under mixed-mode conditions.This stationary phase design can reduce costs and improve work efficiency.Compared with traditional single mode chromatography,MMC has higher selectivity,better resolution and greater sample loading,which make it potential advantages in the separation of complex samples.Reversed-phase/ion-exchange chromatography(RP/IEX)is the most developed MMC attributing to its high orthogonality,and is widely used in the analysis of complex systems.Chromatographic separation depends on the column performance to a large extent.The simplest approach to prepare a column for MMC is by mixing packings with different separation mechanisms.The solute introduced into such a column can interact with the discrete sites independently.However,the resulting mixed-bed columns have poor packing homogeneity and repeatability.Currently,chemical modification of multiple ligands on the stationary phase support is the most commonly used method to prepare MMC.This approach has the advantages of low equipment requirements,high separation selectivity and good packing repeatability.Because of the multiple interactions between ligand and solute,the retention mechanism in MMC has been a complicated issue.Although enhanced retention and selectivity has been demonstrated repeatedly for MMC,many questions concerning the retention mechanisms in MMC remain unanswered and established mixed-mode retention models are debatable.Firstly,the retention of analyte in MMC involves multiple binding interactions,however,cooperative effect arising from the interplay of two or more interactions has been underappreciated.Secondly,there is doubt that cooperative retention in MMC is governed by additivity or cooperativity principle for multiple individual bindings.Finally,there have been no reports on the quantitative assessment of chelate cooperativity in MMC.To solve the above problems,the goals of this work are to prepare RP/SCX and RP/SAX mixed-mode stationary phases through a simple and controllable method,and thoroughly study the cooperative retention mechanism in MMC.In part one,the silica-based mixed-mode RP/SAX and RP/SCX stationary phases were synthesized by a simple two-step procedure.The silica support was first activated with mercaptopropyl silane by vapor-phase deposition,and subsequently functionalized by thiol-ene click reaction with p-vinylbenzyltrimethylammonium chloride or sodium vinylbenzenesulfonate.Then,the prepared stationary phases were evaluated and compared under various chromatographic modes to study their multimodal properties.The results demonstrated that the separation and selectivity of test compounds can be regulated effectively by adjusting various chromatographic conditions,ligand density and ratio.Finally,the mixed-ligand RP/SCX phase was employed for the resolution of melamine and related purities,and the simultaneous separation of acidic,neutral and basic drugs.In part two,the retention mechanism of MMC was studied by comparing the retention behaviors of basic drugs on single-ligand and mixed-ligand RP/SCX stationary phases.The retention factors of selected basic drugs were measured with mobile phase of various pHs,ionic strengths,and solvent strengths.The resultsindicate that the three-interaction-form retention model,containing reversed-phase,pure ion-exchange and cooperative interaction,is more suitable for the mechanism study on MMC.The relative contributions of reversed-phase and ion exchange mechanisms are varied with the solute,mobile phase composition,and ligand type and ratio.Evidence was obtained which showed that hydrophobic and ionic interactions could occur simultaneously with different parts of the solute molecule,giving rise to a significant increase in retention.Moreover,cooperative effects are pronounced on mixed-ligand phase than on its single-ligand counterpart.In part three,a simple method has been developed for quantification of the cooperative effect in MMC.We choose two homologous series of alkylbenzoic acids and alkylanilines as model analytes,and investigated the retention behaviors of the homologs on three bifunctional stationary phases.Cooperative retention was quantified by introducing the definition of cooperative factor.Non-unity values obtained indicate that the retention model in the current system is better described by the cooperativity than additivity principle.Both positive and negative cooperativities have been observed for ionic compounds on the bifunctional RP/IEX stationary phases.Their values remain unchanged with varying hydrophobic and ionic interactions strengths,implying that chelate cooperativity is a structural property,largely determined by the geometric complementarity between interacting ligand and analyte.